Relaxation of smooth muscle of the gut reflects the interplay of nitric oxide (NO), and peptide neurotransmitters, chiefly VIP and its homologue, PACAP. Our previous studies have shown that VIP and PACAP interact with cognate VPAC2 receptors to stimulate cAMP and with the single-transmembrane receptor, NPR-C, to activate smooth muscle eNOS and generate NO and cGMP. Concurrent generation of cAMP and cGMP and activation of PKA and PKG is the physiological norm, and has considerable bearing on the regulation of cyclic nucleotide levels, cyclase and phosphodiesterase (PDE) activities, and the activities of both PKA and PKG. In preliminary studies, we have shown that PKA and/or PKG act upstream to induce feedback desensitization of the VPAC2 and NPR-C receptors, inhibit the synthetic activities of adenylyl and guanylyl cyclases, and augment the degradative activities of cAMP-specific PDE3A and PDE4D5, and cGMP-specific2+ PDE5. Preliminary studies also showed that PKA and/or PKG act on downstream targets to inhibit Ca mobilization and MLC20 phosphorylation, and thus induce relaxation. Characterization of the upstream and downstream molecular targets of PKA and PKG constitutes the main objective of this proposal. The first specific aim is to characterize two novel mechanisms identified in preliminary studies for desensitization of VPAC2 receptors via PKA-specific phosphorylation and potentiation of GRK2, and NPR-C receptors via PKG-specific phosphorylation of a single threonine (Thr 466) in the intracellular domain. The second aim is to characterize the mechanisms that determine the levels of relaxant messengers (NO, cAMP, cGMP), including novel mechanisms of potentiation of PKA-stimulated PDE4D5 by ERK1/2 via inhibition of phosphatase 2A (PP2A), and potentiation of PKG-stimulated PDE5 by PKC via inhibition of PP1, and a mechanism for G((il/i2-mediated activation of eNOS via phosphorylation by PI 3-kinase/Akt. The third aim is to characterize the downstream molecular targets of PKA and/or PKG involved in Ca 2+ mobilization (inhibition of IP3 generation and Ca2+ release via IP 3 and ryanodine receptors; and stimulation of Ca 2+ uptake via SERCA) and sustained MLC20 phosphorylation (inhibition of RhoA and regulators of MLC phosphatase, MYPT1 and CPI-17). Each specific aim is supported by substantial preliminary studies. Their completion should advance our understanding of the molecular mechanisms mediating smooth muscle relaxation.